LESSON 32THEME: MICROORGANISMS CAUSING ZOONOTIC INFECTIONS. BIOLOGICAL PROPERTIES OF MEDICAL IMPORTANT YERSINIA.

CAUSATIVE AGENTS OF PLAGUE, ENTERIC YERSINIOSIS AND PSEUDOTUBERCULOSIS.

MORPHOLOGY AND BIOLOGICAL PROPERTIES OF FRANCISELLA TULARENSIS.

PATHOGENESIS, LABORATORY DIAGNOSTICS AND PROPHYLAXIS OF INFECTIONS.

I. THEORETICAL QUESTIONS

1. General characteristics of the genus Yersinia. Medical important species, their morphology and cultural characteristics.

2. Morphology and cultural characteristics Y. pestis. Virulent factors.3. Morphology and cultural characteristics of Y.enterocolitica and

Y.pseudotuberculosis.4. Biochemical activity and antigen structure of Y.enterocolitica and

Y.pseudotuberculosis.5. Epidemiology and pathogenesis of the plague. Laboratory diagnostics.6. Prophylaxis and treatment of the plague. 7. Epidemiology and pathogenesis of the enteric yersiniosis and

pseudotuberculosis. Laboratory diagnostics of diseases.8. Prophylaxis and treatment of yersiniosis. 9. General characteristics of the genus Francisella. Taxonomic position of F.

tularensis.10. Morphology and cultural characteristics. Antigen structure. Virulent factors.11. Epidemiology and pathogenesis of tularemia. Laboratory diagnostics.12. Prophylaxis and treatment of tularemia.

Yersinia pestis The plague bacillus was discovered independently and simultaneously by Yersin and

Kitasato (1894) in Hong Kong at the beginning of the last pandemic of the disease.Morphology. Y.pestis is a short, plump, ovoid, Gram negative bacillus, about 1.5 μm

x 0.7 μm in size, with rounded ends and convex sides, arranged singly, in short chains or in small groups. In smears stained with Giemsa or methylene blue, it shows bipolar staining (safety pin appearance) with the two ends densely stained and the central area clear. Pleomorphism, is very common and in old cultures, involution forms are seen - coccoid, club shaped, filamentous and giant forms. Pleomorphism is characterically enhanced in media containing 3% NaCl.

Cultural characteristics: The plague bacillus is aerobic_and facultatively anaerobic.Growth occurs over a wide range of pH (pH 5 - 9.6, optimum pH 7.2) and temperature (range 2° - 45°C). The optimum temperature for growth (unlike most pathogens) ) is 27°C but the envelope develops best at 37°C. It is not nutritionally exacting and grows on ordinary media. On nutrient agar, colonies are small, delicate, transparent discs, becoming opaque on continued incubation.

Colonies on blood agar or other hemin containing media are dark brown due to the absorption of the hemin pigment. Colourless colonies are formed on MacConkey agar. In broth, a flocculent growth occurs at the bottom and along the sides of the tube, with little or no turbidity. A delicate pellicle may form later. If grown in a flask of broth with oil or ghee (clarified butter) floated on top (ghee broth) a characteristic growth occurs which hangs

down into the broth from the surface, resembling stalactites (stalactite growth)Biochemical reactions: Glucose, maltose and mannitol but not lactose, sucrose or

rhamnose are fermented with the production of acid but no gas. Indole is not produced. It is MR positive and VP and citrate negative, catalase positive and aesculin positive and oxidase and urease negative. Gelatin is not liquefied. Based on the fermentation of glycerol and reduction of nitrate, Devignat has distinguished three physiological varieties of Y.pestis. This typing appears to be of epidemiological significance be cause of the different geographical distribution of the types.

Resistance: The plague bacillus is easily destroyed by exposure to heat, sunlight, drying and chemical disinfectants. It is destroyed by heat at 55°C or by phenol in 15 minutes. It remains viable for long periods in cold, moist environments. It can survive for several months, and even multiply, in the soil of rodent burrows. All strains are lysed by a specific antiplague bacteriophage at 220C.

Antigens, toxins and other virulence factors:Plague bacilli are antigenically homogeneous and serotypes do not exist. The antigenic

structure is complex. At least 20 antigens have been detected by gel diffusion and biochemical analysis. Many of them have been claimed to be virulence factors.

They include the following: 1. A heat labile protein envelope antigen (FractionI or F-I) best formed in cultures incubated at 37°C. It inhibits phagocytosis and is generally presentonly in virulent strains. This antigen has thereforebeen considered a virulence determinant but occasional strains deficient in Fraction I antigen havebeen isolated from fatal human cases. The antibodyto this antigen is protective in mice. 2. Two antigens designated _V and W and alwaysproduced together have been considered to be thevirulence factors as they inhibit phagocytosis andintracellular killing of the bacillus. Production of Vand W antigens is plasmid mediated. 3. Virulent strains produce a bacteriocin (PesticinI), coagulase and fibrinolysin. Pesticin I inhibitsstrains of Y.pseudotuherculosis ,Y.enterocolitica and E.coli.\

The term 'plague toxins' refers to at least two classes of toxins found in culture filtrates or cell lysates. The first is the endotoxin, a lipopolysaccharide similar to the endotoxins of enteric bacilli. The second class of toxins is protein in nature, possessing some properties of both exotoxins and endotoxins. They are thermolabile and may be toxoided but do not diffuse freely into the medium and are released only by the lysis of the cell. They are called 'murine toxins' as they are active in rats and mice but not in guinea pigs, rabbits and primates. On injection into experimental animals, plague toxins produce local edema and necrosis with systemic effects on the peripheral vascular system and liver. The role of plague toxins in natural disease in human beings is not known.4. Virulence also appears to be associated with an unidentified surface component which absorbs hemin and basic aromatic dyes in culture media to form coloured colonies.5. Virulence has also been associated with the ability for purine synthesis.

Epidemiology: Plague is a zoonotic disease. The plague bacillus is naturally parasitic in rodents. Infection is transmitted among them by rat fleas. The blood, mixed with the bacteria is regurgitated into the bite, transmitting the infection. Infection may also be transferred by contamination of the bite wound with the feces of infected fleas. When a diseased rat dies (rat fall), the fleas leave the carcass and in the absence of another rat, may bite human beings, causing bubonic plague.

In enzootic foci, plague may persist for long periods. Infected fleas may survive for over a year. The bacilli can remain alive and even multiply in the soil of abandoned rodent burrows. They can infect new rodents that may reoccupy such burrows. This may account for the long period of quiescence and subsequent re-emergence characteristic of plague. Attenuated

strains of plague bacilli have been isolated from natural foci. They may regain virulence, when plague becomes active. Eradication of plague is an unlikely prospect as it is a disease of the earth - of rodents that live in burrows and of the fleas that live on them. Only when human beings or domestic animals trespass on these natural foci do human infections set in.

Laboratory diagnosis: The laboratory should be able to diagnose plague not only in human beings but in rodents also, as timely detection of infection in rats may help to prevent epidemic spread.

A rat which died of plague may carry infected fleas and should be handled with care. Pouring kerosene oil over the carcass is a simple method of eliminating the fleas. In the laboratory, the carcasses should be dipped in 3% lysol to destroy ectoparasites.

During epizootics, it is easy to diagnose plague in rats. Buboes are present usually in the cervical region. They are hard and can be moved under the skin. On section, the bubo may show congestion, hemorrhagic points or grey necrosis. Smears from the bubo stained with methylene blue show the bipolar stained bacilli. The fluorescent antibody technique may be of use in identifying plague bacilli in the impression films of the tissues. Bacilli in bubo show considerable pleomorphism. The liver is mottled, with red, yellow or grey stippling. The spleen is enlarged, and moulded over the stomach with granules or nodules on the surface. A characteristic feature is pleural effusion which may be clear, abundant and straw coloured or, less often bloodstained. Bacilli may be demonstrated microscopically in spleen smears and heart blood also. Cultures may be made from the buboes, splee-heart blood and particularly, from bone marrov. decomposed carcasses.

In badly putrified carcasses, microscopy and culture may not be successful. The putrified tissue may be rubbed on the shaven abdomen of a guinea pig. The plague bacillus is able to penetrate through the minute abrasions caused by shaving and initiate lethal infection. Diagnosis in such cases may also be established by the thermoprecipitation test. The tissue, mixed with 5-10 parts of distilled water is boiled for five minutes, filtered and the clear filtrate layered on antiplague serum in a narrow test tube. In positive cases, a precipitate appears at the interface after five minutes incubation at 37°C, increasing to a maximum in two hours.

Diagnosis of sporadic plague in rats may be difficult. Success is achieved by a combination of culture and animal inoculation, using pooled organs. The bacillus may also be isolated from pooled fleas. The only serological test recommended is passive hemagglutination, using tanned sheep erythrocytes sensitised with Fraction I antigen.

In human bubonic plague, a small vesicle may be present at the site of entry of the bacillus in early cases and bacilli may be demonstrated in the vesicle fluid. Bacilli may be readily demonstrated in buboes by microscopy, culture or animal inoculation. Blood cultures are often positive.

In pneumonic plague, the bacilli can be demonstrated in the sputum by microscopy, culture or animal inoculation.

Serological tests are sometimes useful in diagnosis. Antibodies to the F-l antigen may be detected by agglutination or complement fixation tests. The latter test may be used also for detecting the antigen in tissues. Complement fixing antibodies decrease rapidly during convalescence. The passive hemagglutination test, using tanned erythrocytes coated with the F-l antigen or murine toxin is useful for identifying plague foci, as the test remains positive for several years after recovery, from plague.

Prophylaxis: In the prevention of domestic plague, general measures such as control of fleas and rodents are of great importance. Specific protection may be provided by vaccines. Two types of vaccines have been in use - killed and live attenuated vaccines.

The vaccine is given subcutaneously, two doses at an interval of 1-3 months, followed by a third six months later. Vaccination gives some protection against bubonic plague but not against pneumonic plague. The protection does not last for more than six months. In contrast, an attack of plague provides more lasting immunity. A vaccinated person exposed to definite risk of infection should be given chemoprophylaxis - cotrimoxazole or tetracycline orally for at least five

days.The vaccine is recommended only in those exposed occupationally or otherwise to

infections, such as plague laboratory or hospital personnel and troops deployed in known plague areas. It is of no value in plague outbreaks and mass vaccination is not advised. Live plague vaccines cause severe reactions and are not in use now.

Treatment: Early treatment with antibiotics has reduced plague mortality from 30-100 per cent to 5-10 per cent, streptomycin, tetracyclin, chloramphenical and gentamicin are effective.

YERSINIA PSEUDOTUBERCULOSISPseudotuberculosis is a zoonosis transmissible from infected animals to man either

through skin contact with contaminated water or through the consumption of contaminated vegetables or other footis. It occasionally results in a severe, generalized disease with a high fatality rate. More commonly it gives rise to acute mesenteric adenitis, simulating acute or subacute appendicitis (right iliac fossa syndrome), sometimes with the added complication of erythema nodosum, usually in young males (cf. Y. enterocolitica). Enteritis Caused by Y.pseudotuberculosis is rare.

In animals it causes a fatal septicaemia. It attacks wild animals and birds, including rodents, hares and rabbits and also guinea-pigs and other laboratory animals. Subclinical infection may occur. The animal disease must not be confused with so-called pseudotuberculosis of sheep and mice caused by Corynebacterium ovis and C. muris respectively.

Morphology and motility: A small, oval, non-capsulate, Gram-negative, slightly acid-fast, bipolar-stained bacillus. Differs from Y. pestis in being motile at 22°C, readily demonstrated in Craigie tubes incubated at 22°C and at 37°C.

Cultural characteristics: Aerobe and facultative anaerobe: optimum temperature 29°C. Grows slowly on ordinary nutrient agar; colonies are 1 mm in diameter, raised or -umbonate, granular, translucent; non-haemolytic on blood agar; grows poorly on MacConkey medium.

Biochemical activity: Y. pseudotuberculosis strains are biochemically homogenous. They differ from the plague bacillus in their ability to produce urease and from the Pasteurella group in producing β-galactosidase (ONPG positive). Unlike Y. enterocolitica, Y. pseudotuberculosis does not produce ornithine decarboxylase.

Antigenic structure: The strains of Y. pseudotuberculosis are of 6 serological types (serotypes 1-6) based on highly specific somatic antigens, one of which is common to all types, and a thermolabile flagellar antigen (present only in cultures grown at 18-26°C). A rough somatic antigen is shared by all strains and Y. pestis. About 90% of human cases of Y. pseudotuberculosis infections are due to type 1. An antigenic relationship exists between type 2 and type 4 and certain salmonellas of groups B and D respectively. By means of precipitin and haemagglutination tests the strains of Y. pseudotuberculosis has been classified into six O groups (I-VI) on the basis of the O and H antigens so revealed.

Laboratory diagnosis of pseudotuberculosisThe diagnosis is confirmed by isolating the organism from material taken from an

excised mesenteric lymph gland and/or by demonstrating antibodies in the patient's serum during the acute phase of the infection.

Culture. Inoculate the excised material on to nutrient agar, blood agar and MacConkey medium and incubate at 37°C for 18 h. Characteristic colonies are granular, translucent with a beaten-copper surface; on blood agar they are non-haemolytic; on MacConkey medium growth is present but poor.

Prepare films of the colonies and stain by Gram, methylene blue and Ziehl-Neelsen stains. The organisms are small ovoid bacilli, bipolar-stained and weakly acid-fast. In broth

culture at 22°C they are motile, at 37°C non-motile.Serology. Carry out tube agglutination tests with smooth suspensions of strains of types 1-

6 grown at 22°C, either live or inactivated with ethanol or formaldehyde (Report 1983). The tests are incubated at 52°C and read after 4 and 24 h. In positive sera the H agglutinin titers range from 80-12 800. Antibodies decline rapidly. The majority of cases are due to type 1. Since agglutination of types 2 and 4 may result from coagglutinins due to certain types of Salmonella, sera giving these results should be absorbed with salmonellas of groups B and D respectively.

Intradermal test. A skin test similar to the tuberculin and brucellin tests is available to indicate infection by Y. pseudotuberculosis. The reaction may be obtained many years after the infection has cleared.

YERSINIA ENTEROCOLITICA

Y.enterocolitica and related species Y.intermedia, Y. frederikseni and Y. kristenseni constitute a heterologous group of organisms, some of which are parasites and potential pathogens of humans and animals, while others are apparently saprophytic and free-living in water, soil and vegetation. Y. enterocolitica is becoming increase, singly identified as a cause of gastroenteritis in infants and young children and it should be considered in cases of bacillary dysentery or campylobacter-like enterocolitis with abdominal pain and diarrhoea. The incidence is highest during autumn and winter. Occasionally it gives rise to acute terminal ileitis and/or mesenteric lymphadenitis that affects adults of both sexes as well as children. The lesions of this pseudoappendicular syndrome are more severe than those caused by Y. pseudotuberculosis Septicaemia with a high fatality rate among the elderly also occurs but is rare. There may be immunological complications resulting in erythema nodosum, polyarthritis, Reiter's syndrome, etc. The organism has been isolated from many animal species throughout the world but unlike Y. pseudotuberculosis, Y. enterocolitica infections are not considered to be true zoonoses. Human infections probably occur from ingestion or contact. Family and other small outbreaks suggest that person to person transmission occurs.

Morphology and motility: Gram-negative coccobacilli showing pleomorphism in older cultures; apparent capsules are seen in vivo but not in culture; motile by means of peritrichous flagella when grown at 22°C, non-motile at 37°C.

Cultural characteristics: Aerobe and facultative anaerobe. Optimum temperature 22-29°C; multiplies at 4°C (which constitutes a hazard when contaminated food is refrigerated). They grow slowly on artificial media; on blood agar forms non-haemolytic, smooth, translucent colonies. 2-3 mm in diameter in 48 h; grows on MacConkey medium forming pinpoint colonies at 22°C. Selective media or enrichment techniques are necessary for isolation from faecal specimens.

Sensitivity: Like other yersinias. Y. enterocolitica is killed by heat at 55°C and by phenol (0.5%) in 15 min. It is susceptible to sulphadiazine, streptomycin, tetracycline and chloramphenicol, but not to penicillin. Antibiotics should be used only for the treatment of severe or generalized infections in adults: cotrimoxazole is effective.

Biochemical activity: Y. enterocolitica is more reactive at 28°C than at 37°C. It differs from Y. pseudotuberculosis in its ability to ferment sucrose, sorbitol, cellobiose but not salicin, and in being ornithine decarboxylase positive and Voges-Proskauer positive. On the basis of variations in certain biochemical tests Y. enterocolitica may be divided into six different biotypes and three new species are now recognized, viz. Y. intermedia and Y. frederikseni which differ from it in their ability to ferment rhamnose and Y. kristenseni which does not ferment sucrose.

Toxin production: Pathogenic serotypes produce a heat-stable enterotoxin similar to that

produced by entero-toxigenic Escherkhia coli. Because the toxin is not produced at temperatures exceeding 30°C it is unlikely that it contributes to the pathogenicity of the infecting strain. However the toxin may be developed by organisms growing in contaminated food stored at low temperatures. This may explain the occurrence of some cases of food poisoning from which no causative organism has been isolated. Toxigenic strains do not ferment rhamnose.

Antigenic structure: Y. enterocolitica is divisible into a large number of serotypes depending on 34 different О antigen factors and 19 H factors. Serotypes 3 and 9 account for the majority of human infections, especially in Europe; in the USA serotype 8 is more common. Other serotypes not associated with human disease have been isolated from healthy individuals and from milk, meat and vegetables. They are probably non-pathogenic. Serotype 9 may cross-react with some Brucella species.

Laboratory diagnosis of Y.enterocolitica infectionsThe diagnosis is confirmed directly by isolation of the organism and indirectly by

serological tests on the patient's serum.

Isolation from blood or lymph nodes: Inoculate on to blood agar and MacConkey agar. Incubate plates at 22-29°C for 24 h.

Isolation from faeces, food, soil, etc.1. Subject heavily contaminated material to preliminary enrichment by mixing with

phosphate buffered saline or peptone water, pH 7.6. Maintain at 4°C for 22 days or more.2. Subculture at weekly intervals on a selective medium or on MacConkey medium

containing a minimum amount of bile salt. Incubate at 22-29°C. Examine plates for colonies of Y. enterocolitica which on the selective medium appear like a bulls eye, coloured dark red and surrounded by a transparent border. The size varies according to the serotype of the strain. Other organisms that grow produce larger colonies with pinkish centers.

3. Prepare pure cultures; test for motility at22°C; identify the strain by biochemical tests.

4. Determine the serotype of the strain by slide agglutination against rabbit antisera to Y. enterocolitica serotypes 3 and 9 using factor О and OH antisera. If serotype 3, subdivide by phage typing.

Serological diagnosis: Use О antigen preparations of serotypes 3 and 9 to test the patient's serum at time of onset of illness and 10 days later by tube agglutination test. A rising titer of 160 and over is significant.

F.tularensisF.tularensis is the cause of tularaemia, a plague-like disease of rodents and

other small mammals. It is tick-borne among the natural hosts and transmissible to man as a typical zoonosis, either through direct or indirect contact with infected animals, or through handling laboratory cultures without strict safety precautions. The disease is widespread in North America but in Europe it is limited to certain countries and has not yet been identified in the UK. The so-called 'lemming fever' in Norway results from the consumption of water polluted with the classes or excreta of infected lemmings or water rats. Water-borne or airborne infections tend to produce influenza-like, pulmonary

or typhoid-like illnesses, but in persons such as butchers or trappers who become infected through handling the tissues of animals such as rabbits or hares, there may be ulceroglandular or oculoglandular manifestations

Morphology: Small coccobacillus which on primary isolation does not exceed 0.7 x 0.2 μm; in culture pleomorphic. capsulate, non-motile and non-sporing; Grarn-negative showing bipolar staining; stains best with dilute (10%) carbol fuchsin as counter-stain. Smears from post-mortem material may require gentle heating to allow penetration of the stain.

Cultural characteristics: Strict aerobe. Fresh isolates cannot be cultured on ordinary medium but require a complex medium confining blood or tissue extracts and cystine. Optimum temperature is 37°C. Minute droplet-like colonies develop in 72 h. Growth in liquid culture medium may be obtained using casein hydrolysate with added thiamine and cystine.

Sensitivity: Killed by moist heat at 55-60°C in 10 min. May remain viable for many years in cultures kept at 10°C and in humid soil and water for 30 and 90 days respectively. It is very sensitive to chlorampheficol; sensitive to streptomycin. Tetracycline is bacteriostatic and only effective in large doses, 2 g/day for 14 days; it is used for prophylaxis and therapy when the infecting strain is streptomycin resistant.

Biochemical activity: Under suitable conditions acid is formed from glucose and maltose. Indole and urease tests are negative.

Laboratory diagnosisIsolate and identify the organism from material taken from the lesions, and

demonstrate specific antibodies in the patient's serum.1. Culture the discharge from local lesionsor glands on special medium, e.g.

blood agar enriched with 0.1% cystine, or inspissated egg yolk medium. Incubate in air with 10% CO: at 37°C for 72 h or more. Small mucoid colonies are characteristic.

2. Inoculate exudates from ulcers and glands into guinea-pigs and mice. Culture the liver and spleen of the infected animals post mortem on special medium.

3. Obtain pure cultures for identification of F.tularensis.4. Perform slide agglutination tests on animal serum and fluorescent antibody

tests on spleen imprints.5. Test patient's serum for specific antibodies by slide and tube agglutination

tests, haemag-glutination, complement fixation and anti-human globulin (Coombs) test (see Chs 10 and 33), using antigens prepared from suspensions of F. tularensis grown on solid medium). Serum of cases of brucellosis may cross-react with F. tularensis. Diagnostic antigens and antisera for use in slide and tube agglutination tests are available from Difco.

II Students practical activities

1. Prepare smear from pure culture of Y.pseudotuberculosis and Y.enterocolitica, stain by Gram and microscopy. Find the bacteria and sketch the image.

1. Microscopy the prepared smears from pure culture of Y. pestis stained by Gram and with methylene blue. Estimate the morphology and sketch the image.

2. Microscopy the prepared smears from pure culture of F. tularensis stained by Gram. Sketch the image.

3. To estimate the cultural characteristics of Y.pseudotuberculosis and Y.enterocolitica, have been grown on the nutrient agar.

2. Familiarize with specific media for cultivation of Francisella tularensis.

3. Detect the biochemical features of Y.enterocolitica and Y.pseudotuberculosis . Note them in protocol.

4. Write down the principal scheme of laboratory diagnostics of plague and tularemia.